Federico Di Gesualdo

Glucose availability in hypoxia regulates the selection of chronic myeloid leukemia progenitor subsets with different resistance to imatinib-mesylate

Background Incubation of chronic myeloid leukemia cells in hypoxia inhibits growth and selects BCR/Abl-independent cells with stem cell properties which are refractory to imatinib-mesylate. This study aimed to characterize the relationship of this refractoriness with glucose availability in the environment. Design and Methods K562 or primary chronic myeloid leukemia cells were cultured at 0.1% O2, different cell densities and glucose concentrations. The stem and progenitor cell potential of these cultures at different times of incubation in relation to BCR/Ablprotein expression and sensitivity to imatinib-mesylate was explored by transferring cells to growth-permissive secondary cultures in normoxia, according to the Culture-Repopulating Ability assay methodology. Results Hypoxia-resistant cells maintained BCR/Ablprotein expression until glucose was no longer available in primary hypoxic cultures, where glucose availability appeared to regulate cell number and the balance between the enrichment of cells with kinetic properties typical of stem or progenitor cells. Cells surviving merely hypoxic conditions were, upon transfer to secondary cultures, immediately available for numerical expansion due to the maintained BCR/Ablprotein expression, and were consequently sensitive to imatinib-mesylate. Instead, BCR/Ablprotein–negative cells selected in primary cultures under oxygen/glucose shortage underwent a delayed numerical expansion in secondary cultures, which was completely refractory to imatinib-mesylate. Cells with the latter properties were also found in primary chronic myeloid leukemia explants. Conclusions Glucose shortage in hypoxia was shown to represent the condition selecting BCR/Ablprotein–negative cells refractory to imatinib-mesylate from either chronic myeloid leukemia lines or patients. These cells, exhibiting stem cell properties in vitro, are metabolically suited to home to stem cell niches in vivo and so may represent the chronic myeloid leukemia cell subset responsible for minimal residual disease.

ζ-crystallin is a bcl-2 mRNA binding protein involved in bcl-2 overexpression in T-cell acute lymphocytic leukemia

The human antiapoptotic bcl‐2 gene has been discovered in t(14;18) B‐cell leukemias/lymphomas because of its overexpression caused at a transcriptional control level by the bcl‐2/IgH fusion gene. We were the first to disclose the post‐transcriptional control of bcl‐2 expression mediated by interactions of an adenine + uracil (AU)‐rich element (ARE) in the 3′‐UTR of bcl‐2 mRNA with AU‐binding proteins (AUBPs). Here, we identify and characterize ζ‐crystallin as a new bcl‐2 AUBP, whose silencing or overexpression has impact on bcl‐2 mRNA stability. An increased Bcl‐2 level observed in normal phytohemagglutinin (PHA)‐activated T lymphocytes, acute lymphatic leukemia (ALL) T‐cell lines, and T cells of patients with leukemia in comparison with normal non‐PHA‐activated T lymphocytes was concomitant with an increase in ζ‐crystallin level. The specific association of ζ‐crystallin with the bcl‐2 ARE was significantly enhanced in T cells of patients with ALL, which accounts for the higher stability of bcl‐2 mRNA and suggests a possible contribution of ζ‐crystallin to bcl‐2 overexpression occurring in this leukemia.—Lapucci, A., Lulli, M., Amedei, A, Papucci, L., Witort, E., Di Gesualdo, F., Bertolini, F., Brewer, G., Nicolin, A., Bevilacqua, A., Schiavone, N., Morello, D., Donnini, M., Capaccioli, S. ζ‐Crystallin is a bcl‐2 mRNA binding protein involved in bcl‐2 overexpression in T‐cell acute lymphocytic leukemia. FASEB J. 24, 1852–1865 (2010). www.fasebj.org

Different BCR/Abl protein suppression patterns as a converging trait of chronic myeloid leukemia cell adaptation to energy restriction

BCR/Abl protein drives the onset and progression of Chronic Myeloid Leukemia (CML). We previously showed that BCR/Abl protein is suppressed in low oxygen, where viable cells retain stem cell potential. This study addressed the regulation of BCR/Abl protein expression under oxygen or glucose shortage, characteristic of the in vivo environment where cells resistant to tyrosine kinase inhibitors (TKi) persist. We investigated, at transcriptional, translational and post-translational level, the mechanisms involved in BCR/Abl suppression in K562 and KCL22 CML cells. BCR/abl mRNA steady-state analysis and ChIP-qPCR on BCR promoter revealed that BCR/abl transcriptional activity is reduced in K562 cells under oxygen shortage. The SUnSET assay showed an overall reduction of protein synthesis under oxygen/glucose shortage in both cell lines. However, only low oxygen decreased polysome-associated BCR/abl mRNA significantly in KCL22 cells, suggesting a decreased BCR/Abl translation. The proteasome inhibitor MG132 or the pan-caspase inhibitor z-VAD-fmk extended BCR/Abl expression under oxygen/glucose shortage in K562 cells. Glucose shortage induced autophagy-dependent BCR/Abl protein degradation in KCL22 cells. Overall, our results showed that energy restriction induces different cell-specific BCR/Abl protein suppression patterns, which represent a converging route to TKi-resistance of CML cells. Thus, the interference with BCR/Abl expression in environment-adapted CML cells may become a useful implement to current therapy.